Thyroxine-binding globulin (TBG) was purified from human plasma and shown to have a molecular weight of 54000 and a carbohydrate content of 23%. Quantitative C-terminal amino acid analysis, peptide mapping and amino acid compoistion, and denaturation of the reduced, alkylated protein showed that TBG is a single polypeptide chain. The molecule has a single binding site for the hormone, which is irreversibly lost when TBG is acidified below pH 5. TBG is a compact, symmetric molecule containing about 25% alpha-helical and 25% Beta-structures. The binding of thyroid hormone analogs by serum prealbumin (PA) was studied by a fluorescence method employing ANS. Analysis of binding required two models, since some analogs, but not others, caused quenching of ANS fluorescence while the chromophore occupied one of the two binding sites on PA. The thyroxine analog 3,5-dinitro-3',5'-diiodothyroproprionic acid was synthesized. Its suitability as a photoaffinity label for the thyroxine-binding site on prealbumin was investigated.